Vesicles comprising epidermal growth factor and compositions thereof

ABSTRACT

The invention relates to vesicles comprising Epidermal Growth Factor (EGF), a cationic surfactant and cholesterol or derivatives thereof. The invention also discloses a procedure for their preparation, based on compressed fluid technology (CFs). The vesicles of the invention are useful in the manufacture of drugs and cosmetics and in tissue engineering.

This application is the U.S. National Phase of, and Applicant claimspriority from, International Patent Application Number PCT/CU2013/000004filed Aug. 2, 2013, which claims priority from CU 2012-0112 filed Aug.2, 2012, each of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention falls within the fields of human and veterinarymedicine, cosmetics and tissue engineering, particularly in the field ofvesicular type release systems comprising Epidermal Growth Factor (EGF)in their composition. The vesicles of the invention have improvedtherapeutic efficacy in relation to free EGF.

PRIOR STATE OF THE ART

Drug delivery systems based on vesicular systems, usually liposomes,composed of amphiphilic molecules that incorporate a therapeuticallyactive substance, constitute one of the most widely used systems in thepharmaceutical sector, because they can provide increased stability tothe active principle, increase its permeability through biologicalmembranes, and allow slow release of the active ingredient without theneed of repeated administrations.

EGF is one of the main growth factors that stimulate cell proliferationand motility during tissue regeneration. It also helps maintain tissuehemostasis through the regulation of epithelial cell proliferation andmigration. Furthermore, EGF induces angiogenesis, which providesnutritional support to the tissue (Hudson and McCawley, Microsc. Res.Tech. 1998, 43: 444-455; Koivisto et al., Exp. Cell Res. 2006, 312:2791-2805; Liang et al., Wound Repair Regen. 2008, 16: 691-698). Thisgrowth factor has multiple applications in the pharmaceutical field(Wong et al., Biotechnol. Genet. Eng. Rev. 2001, 18: 51-71; Girdler etal., Am. J. Clin. Oncol. 1995, 18: 403-406; Haedo et al., Rev. Esp.Enferm. Dig. 1996, 88: 409-413; Majima, Ophthalmologica 1998,212:250-256); in cosmetics (Hasegawa and Yamamoto, Mech. Ageing. Dev.1992, 66:107-114, U.S. Pat. No. 5,618,544) and in tissue engineering(Christopher et al., Biomacromolecules 2011, 12: 3139-3146).

EGF formulations using different liposome systems have been developed.Some examples are EGF integration in unilamellar liposomes comprisingphosphatidylglycerol (PG), phosphatidylcholine (PC) and cholesterol(Brown et al., Ann. Surg. 1988, 208: 788-794). The inclusion of EGF hasalso been reported in multilamellar liposomes comprising PC, cholesteroland hyaluronic acid (Yerushalmi, et al., Arch. Biochem. Biophys. 1994,313: 267-273); or comprising cholesterol anddipalmitoylphosphatidylcholine (DPPC) (Alemdaro{hacek over (g)}lu etal., J. Biomed. Mater. Res. A 2008, 85A: 271-283; De{hacek over (g)}imet al., Int. Wound. J. 2011, 8: 343-354). Another class of liposomesthat have been reported are the multivesicular ones comprisingdioleoylphosphatidylcholine (DOPC), dimyristoyl phosphatidylglycerol(DMPG), cholesterol and triolein (Li et al., Arch. Pharm. Res. 2005, 28:988-994).

On the other hand, liposomes that comprise cationic lipids conjugated toEGF have been reported (Kikuchi et al., Biochem. Biophys. Res. Commun.1996, 227: 666-671); polyethylene glycol (PEG) coated liposomes, whichalso comprise cholesterol and dioleoylphosphatidylethanolamine (DOPE) incombination with PC or DPPC (Li et al., Int. J. Pharm. 2003, 258:11-19). Liposomes comprising DPPC and lysophosphatidylcholine (LPC) havealso been reported (Saddi et al., The Angle Orthodontist 2008, 78:604-609; Alves et al., Life Sci. 2009, 85: 693-699).

EGF formulations using liposomal systems have also been protected bypatents, such as the one disclosing the gel composition of EGF/liposomeand methods comprising EGF entrapment in liposomes containing neutraland negatively-charged phospholipids (U.S. Pat. No. 4,944,948); the gelcomposition of liposomes and methods that use negatively-chargedliposomes and EGF, and include negatively charged lipids, such as: PG,PC and cholesterol (International Patent Application No. WO 9009782).Another patent application relates to the topical application of EGF inliposomes to prevent diabetic foot amputation, and liposomes comprisingPC and sodium deoxycholate are used. This patent application isrestricted to the use of any type of liposomes/niosomes of EGF for thetopical treatment of grades IV and V chronic ischemic lesions ofdiabetic foot (International Patent Application No. WO 2007/073704). Inthe prior art, EGF integration in vesicular systems constituted bycholesterol and cationic surfactants has not been found.

Traditional methods for obtaining liposomes, such as thin-filmevaporation (Agrawal et al., J. Liposome Res. 2005, 15: 141-155),dehydration-rehydration (Kirby and Gregoriadis, Nat. Biotechnol. 1984,2: 979-984), freeze-thawing (Ristori et al., Biophys. J. 2005, 88:535-547) and extrusion (MacDonald et al., Biochim. Biophys. Acta 1991,1061: 297-303) have certain drawbacks. Some of these drawbacks areassociated to the use of large amounts of solvents, which are difficultto eliminate afterwards, or with the high temperatures required by someof these methods, limiting their use to thermally stable substances. Onthe other hand, the size and nanostructuring of the material aredifficult to control and these methods have low reproducibility duringthe scale-up (multi-stage processes). Another problem with liposomepreparations is their poor stability.

Processing of materials with compressed fluids (CFs), or dense gases,both in liquid or supercritical state, as solvents, have aroused greatexpectancy at academic and industrial levels, for preparing micro- ornanostructured materials such as: particulate materials, vesicularsystems, composite particles, structured surfaces, etc., with greaterstructural homogeneity than that achieved by conventional processing(Holmes et al., Chem. Eur. J. 2003, 9: 2144-2150; Cooper, Adv. Mater.2001, 13: 1111-1114; Cooper, Adv. Mater. 2003, 15: 1049-1059 and Woodset al., J. Mater. Chem. 2004, 14: 1663-1678). A CF or a dense gas is asubstance that at normal conditions of pressure and temperature existsas gas but increasing the pressure can be converted into liquids orsupercritical fluids, and be used as solvent media for chemical andmaterial processing. The most frequently used CF is carbon dioxide(CO₂), classified as green solvent, because it is non-toxic,non-flammable, easy to remove, leaves no residues in the particles, isinexpensive and easy to recover. Since the early '90s, a series ofmethodologies that use CFs for preparing finely divided materials, withmicro-, sub-micro and nanoscopic particle sizes, have been developed(Jung and Perrut, J. Supercrit. Fluid, 2001, 20: 179-219). The solvatingpower of CFs may be modified by temperature and composition changes, asin the case of conventional liquid solvents, and also by pressurechanges, which are transmitted much faster within solutions. Therefore,these precipitation methods have in common the possibility of achievingvery high grades of supersaturation in very short time intervals,promoting nucleation over crystal growth and thereby obtaining micro- ornanoparticles with very narrow size distributions, controlled internalstructure and supramolecular organization.

One of the processes to obtain micro- or nanostructured materials withCFs is the method called DELOS-SUSP—Depressurization of an ExpandedLiquid Organic Solution-Suspension—(International Patent Application No.WO 2006/079889; Patent No. EP 1843836; Cano-Sarabia et al., Langmuir2008, 24: 2433-2437), which is based on depressurization of an organicsolution previously expanded by a CF, generating either a micro- or ananodisperse system by said depressurization. In this process, the CFacts as co-solvent, being completely miscible, under certain conditionsof pressure and temperature, with the organic solution of the solute tobe stabilized as micro- or nanodisperse system. Said stabilization isachieved in the presence of additives in the medium, usually aqueous, onwhich depressurization of the expanded solution is carried out.Additives may be emulsifiers, ionic and non-ionic detergents, surfaceagents, colloid stabilizers and protectors. Using this method micro-and/or nanodisperse systems, such as liposomes, emulsions orsuspensions, can be obtained. The liposomes or vesicles are composed ofcholesterol and other membrane agents, such as phospholipids andsurfactants, and their preparation requires dissolution of cholesteroland/or other lipids in the expanded organic solution and itsdepressurization in an aqueous surfactant solution.

For the possible incorporation of actives in vesicles or liposomes byDELOS-SUSP, and to generate the corresponding vesicles, it is requiredto dissolve the active ingredient in the initial expanded solution, orin the aqueous solution, in which depressurization of said expandedsolution is carried out and in both cases, this dissolution must becarried out in the presence of lipids, detergents or surface activeagents.

Among cationic surfactants, those of quaternary ammonium type (QUATs)have been widely used in pharmaceuticals and cosmetics. In thepharmaceutical field, they have been used by topical, ophthalmic, oral,buccal and nasal routes. Previously, the preparation ofcholesterol:cetyltrimethylammonium bromide (CTAB) nanovesicles usingDELOS-SUSP technology has been reported. One example of theincorporation of water soluble compounds by this technology is describedin the reference “Liposomes and other vesicular systems: structuralcharacteristics, methods of preparation, and use in nanomedicine”(Progress in Molecular Biology and Translational Science, Elsevier,2011, vol. 104, pp. 1-52), where cholesterol:CTAB vesicles are used asvehicle for encapsulation and administration of the antibioticgentamicin. It must be emphasized that reported gentamicinencapsulations are very low (<2%). This type of vehicle has never beenused to incorporate proteins. It is known that ionic detergents areagents that cause protein denaturation (Akin et al., Anal. Biochem.1985, 145: 170-176; Andersen et al., J. Mol. Biol. 2009, 391: 207-226).Denaturation of human serum albumin after CTAB addition has beenrecently demonstrated by Raman spectroscopy (Vlasova and Saletsky, LaserPhys. 2011, 21: 239-244). Denaturation, as a rule, is accompanied by theloss of functional properties of the protein.

Due to all the above mentioned reasons, it is still of interest toachieve new release systems of EGF, easy to standardize, with highhomogeneity at the structural level and in its physico-chemicalproperties, that improve the pharmaceutical and pharmacologicalproperties and/or increase the therapeutic activity of EGF.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to vesicles, as drug delivery system,which comprise EGF, a cationic surfactant and cholesterol or one of itsderivatives and have greater therapeutic effectiveness than thosepreviously described.

The present invention also relates to a process for the preparation ofsaid vesicles comprising EGF, a cationic surfactant and cholesterol orderivatives thereof, that includes: a) preparation of an aqueoussolution of EGF and a cationic surfactant, b) the dissolution ofcholesterol or one of its derivatives in an organic solvent expandedwith a CF, c) the synthesis of the vesicles by depressurization of thesolution resulting from stage b) on the solution resulting from stagea).

A pharmaceutical composition characterized by comprising vesicles thatinclude EGF, a cationic surfactant and cholesterol, or derivativesthereof, and at least one pharmaceutically acceptable excipient is alsoan object of the invention. Another object of the invention is the useof said vesicles for the manufacture of medicaments and cosmetics.

The pharmaceutical compositions of this invention, which contain EGFvesicles with other components, are useful as drugs to accelerate thehealing process of diabetic foot ulcers and other complex wounds, suchas: venous ulcers, decubitus ulcers, burns, among others; to repair theanterior chamber structures in damaged eyes, in systemic mucositis andin all the diseases of the gastrointestinal tract that involve the needto regenerate mucosa and submucosa. In particular, it has been foundthat these vesicles have significantly greater therapeutic effectivenessfor healing diabetic foot ulcers and venous ulcers than those describedin the prior art.

The invention also relates to a cosmetic product characterized bycomprising vesicles of EGF, a cationic surfactant and cholesterol orderivatives thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of an equipment for obtaining vesiclescomprising EGF, a cationic surfactant and cholesterol or acholesterol-derivative where: C, Collector; H, Heat exchanger; P, Pump;R, Reactor; V, Valve; RD, Rupture disc; ST, Stirrer; FL, Filter; TI,Temperature indicator; PI, Pressure indicator; PIC, Pressure indicatorcontroller; F, Flow meter.

FIG. 2. Particle size distribution by dynamic light scattering (DLS) ofthe vesicles keeping a constant CTAB:cholesterol ratio at 1 M:1 M andvarying the EGF:cholesterol ratio (0 μM:1 M (

), 5 μM:1 M (

), 15 μM:1 M (

), 25 μM:1 M (

) and 40 μM:1 M (

) (A); and keeping the tetradecyl methylammonium bromide(cetrimide):cholesterol relation constant at 1 M:1 M and varying theEGF: cholesterol ratio (0 μM:1M (

), 5 μM:1 M (

), 15 μM:1 M (

), 25 μM:1 M (

) and 40 μM:1 M (

)(B).

FIG. 3. Pictures of cryo-transmission electron microscopy (Cryo-TEM) ofEGF vesicles with cholesterol:CTAB:EGF composition (A),cholesterol:cetrimide:EGF (B), cholesterol:benzalkonium chloride(BKC):EGF composition (C) and β-Sitosterol:CTAB:EGF (D) withQUATs:cholesterol or β-Sitosterol ratio of 1 M:1 M and EGF:cholesterolor β-Sitosterol ratio of 5 μM:1 M.

FIG. 4. Specific biological activity of different EGF preparations in acell proliferation assay, where free EGF, DPPC:cholesterol liposomes(with ratio DPPC:cholesterol 1 M:1 M and EGF:cholesterol 25 μM:1 M) andthe CTAB:cholesterol vesicles (A) and cetrimide:cholesterol vesicles(B), keeping the QUATs:cholesterol 1M:1M ratio constant and varying theEGF:cholesterol (5 μM:1M, 15 μM:1M and 25 μM:1M) ratio are compared.

FIG. 5. Proteolytic degradation profile, after exposure to trypsin at37° C. during different time intervals, of free EGF and differentvesicle preparations keeping constant the CTAB:cholesterol ratio (A) andthe cetrimide:cholesterol ratio (B) at 1 M:1 M and varying theEGF:cholesterol ratio (5 μM:1 M, 15 μM:1 M and 25 μM:1 M).

FIG. 6. Photographs of the healing evolution of the diabetic foot ulcercorresponding to patient JLG at treatment onset (A), after 4 weeks (B)and 8 weeks (C) of treatment with a topical spray formulation containingvesicles with CTAB:cholesterol ratio 1 M:1 M and EGF:cholesterol ratio 5μM:1 M, with an EGF equivalent concentration of 15 μg/mL.

FIG. 7. Photographs of the healing evolution of the diabetic foot ulcercorresponding to patient ZEM at treatment onset (A), after 4 weeks (B)and 8 weeks (C) of treatment. During the first 4 weeks the treatment wascarried out by infiltration with a parenteral formulation containingvesicles with BKC:cholesterol ratio 1 M:1 M and EGF:cholesterol ratio 5μM:1 M, with an equivalent concentration of EGF of 75 μg/mL. The other 4weeks, to complete 8 weeks, the treatment was carried out by using atopical spray formulation containing vesicles with CTAB:cholesterolratio 1 M:1 M and EGF:cholesterol ratio 5 μM:1 M, with an equivalentconcentration of EGF of 15 μg/mL.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides vesicles that are characterized bycomprising epidermal growth factor (EGF), a cationic surfactant andcholesterol or derivatives thereof. In an embodiment of the invention,the cationic surfactant is of quaternary ammonium type.

In the context of this invention the term “EGF” refers to any variant ofthe EGF molecules that maintains their biological activity; for example,C-terminal truncated molecules (Calnan et al., Gut 2000, 47: 622-627);or molecules truncated at the N-terminal (Svodoca et. al., Biochim.Biophys. Acta 1994, 1206: 35-41; Shin et al., Peptides 1995, 16:205-210). EGF can be obtained by recombinant DNA technology using yeastslike Saccharomyces (Valdés et al., Biotecnol. Apl. 2009, 26: 1-9) orPichia pastoris (Research Journal of International Studies 2009, 10:36-46); using bacteria, such as Escherichia coli (Yoon et al.,Biotechnol. Bioprocess Eng. 1997, 2: 86-89; Abdull Razis et al., Appl.Biochem. Biotechnol. 2008, 144: 249-261); or by methods of chemicalsynthesis (Shin et al., Peptides 1995, 16: 205-210). The EGF object ofthe invention also comprises whichever variant obtained by thepreviously described methods, after being modified by any procedure ofthe prior art, such as: amino acid substitution (Shiah et al., J. Biol.Chem. 1992, 267: 24034-24040; Lahti et al., FEBS Lett. 2011, 585:1135-1139; International Patent Application No. WO 2007/065464), andpolyethylene glycol conjugation (Thomas et al., Bioconjugate Chem. 2001,12: 529-537; Lee et al., Pharm. Res. 2003, 20: 818-825), or any othermethod of chemical or genetic modification.

The term “cationic surfactant” refers to those surfactants with at leastone positive charge in the molecule and also includes the combination ofone or more cationic surfactants. For example, according to the presentinvention, cationic surfactants of the tertiary amine salt type,quaternary ammonium salt and alkyl ammonium in saturated and unsaturatedheterocycles can be used.

In the invention, the term “quaternary ammonium type (QUATs) cationicsurfactant” refers to quaternary ammonium salts in which at least onenitrogen substituent is a long chain. Compounds such as CTAB, cetrimideand BKC or their mixture are included among QUATs. In a preferredembodiment of the present invention, the cationic surfactant used is asurfactant acceptable in pharmaceutics. The QUATs as well as the rest ofthe cationic surfactants can be obtained from commercially availablesources, with pharmaceutical and cosmetic qualities.

In the present invention, the term “vesicles” refers to colloidalmicroparticles and nanoparticles, which are between 25 nm and 5 μm andare formed by one or more bilayers of amphiphilic molecules that containan aqueous phase.

In one embodiment of the invention, the vesicles have a molar ratio ofcationic surfactant to cholesterol (or derivatives thereof) in the rangeof 10 M:1 M to 1 M:5 M and a molar ratio of EGF to cholesterol (or itsderivatives thereof) which is in the range of 0.5 μM:1 M to 100 μM:1 M.

The term “derivatives” of cholesterol, in the present invention, refersto molecules of the steroids family, generally obtained from thecholesterol precursor molecule and having lipophilic character.

In one embodiment of the invention, the vesicles comprising EGF arecharacterized by having unilamellar structure and approximate mean sizebetween 25 and 500 nm, preferably between 50 and 300 nm. In a particularembodiment, the invention refers to vesicles in which EGF isincorporated into the vesicle bilayer. The approximate size andmorphology of the vesicles are evaluated by Cryo-TEM and thedistribution of vesicle size is characterized by DLS.

Surprisingly, the vesicles of the invention show significant increase inthe biological potency of EGF (measured in vitro) compared to free EGFand EGF in cholesterol:DPPC liposomes. Moreover, these vesicles arecapable of protecting EGF against protease attack, a very importantcharacteristic for achieving adequate bioavailability of EGF at the siteof action; therefore, increasing its therapeutic effectiveness.

In the present invention, for the first time, EGF vesicles that improvesome of the pharmaceutical and pharmacological properties of this growthfactor, such as its potency and stability, have been synthesized. It hasbeen demonstrated that the degree of incorporation of EGF in thestructure of the vesicles remains stable for at least one year.Additionally, they enable to increase of permeability through biologicalmembranes.

The vesicles of the invention have the additional advantage ofantimicrobial and antifungal effect, which is desirable in compositionsused in the treatment of complex wounds and other lesions susceptible ofEGF treatment.

In an embodiment of the invention, the vesicles comprising EGF areobtained by CF technology. In a particular embodiment, the CF technologythat is used to obtain the vesicles includes a process comprising a)preparation of an aqueous solution of EGF and a cationic surfactant, b)dissolution of cholesterol or derivatives thereof in an organic solventexpanded with a CF, and c) vesicle synthesis by depressurization of thesolution resulting from stage b) on the solutions resulting from stagea). In a preferred embodiment, the cationic surfactant used in stage a)is of quaternary ammonium-type.

The invention also provides a process for preparing vesicles thatcomprise EGF, a cationic surfactant and cholesterol or its derivativescharacterized by comprising a) the preparation of an aqueous solution ofEGF and a cationic surfactant, b) dissolution of cholesterol orderivatives thereof in an organic solvent expanded with a CF, and c)vesicle synthesis by depressurization of the resulting solution in stageb) on the solution resulting in stage a). In one embodiment of theinvention, the above described process includes a cationic surfactant ofquaternary ammonium type.

In one embodiment of the invention, the organic solvent in stage b) ofthe above mentioned process is a solvent selected from the group formedby monohydric alcohols, such as: ethanol, methanol, 1-propanolol,2-propanolol, 1-butanol, 1-hexanol, 1-octanol and trifluoroethanol;polyhydric alcohols, such as: propylene glycol, PEG 400 and1,3-propanediol; ketones, such as acetone, methyl ethyl ketone andmethyl isobutyl ketone; ethylenediamine, acetonitrile, ethyl acetate andmixtures thereof. In any case, whatever is the nature of the organicsolvent, the lipid component has to be soluble in it and further, saidsolvent has to necessarily be miscible in CF and water. Moreover, theselected organic solvent must have relatively low toxicity.

The relative concentration of EGF and surfactant in the initial buffersolution and the cholesterol concentration in the organic solvent aredetermined by the desired ratio of cholesterol:cationic surfactant:EGFin the final vesicle. In general, the cholesterol: cationicsurfactant:EGF ratio can influence the physico-chemical and biologicalproperties of the different vesicles obtained.

In another embodiment of the invention, the CF used in the processdescribed before is a component selected between CO₂, ethane, propane,hydrochlorofluorocarbons (eg., CFC-22), and hydrofluorocarbons (eg.,HFC-134A). Preferably, the CF in stage b) is CO₂, considered anecological solvent, because it is non-toxic, non-flammable,non-corrosive, is not harmful for the environment and moreover, is veryabundant in nature.

In one embodiment of the invention, the process for EGF vesiclepreparation is performed in an apparatus, as the one shown in FIG. 1. Itconsists of a high-pressure reactor (R) to which a solution ofcholesterol in ethanol at concentration (C₁) is added at atmosphericpressure and working temperature (T=T_(w)). In a second stage,compressed CO₂ is added until the working pressure (P=P_(w)) is reached,producing volumetric expansion of the solution to a molar fractionX_(CO2). The addition is done through valve V-1 using pump P1, keepingthe rest of the valves closed. The system is maintained at pressureP_(W) and temperature T_(W), during a specified time, to ensure completehomogenization and thermal balance. After this time, V-4 is opened toconnect reactor R to filter FL, which has previously been pressurizedwith N₂ to P_(W) keeping the rest of the valves closed. Opening V-6allows depressurization of the volumetrically expanded solution on anaqueous EGF solution at concentration (C₂) and surfactant atconcentration (C₃) pumped through P2. In this final stage, a stream ofN₂, added through V-2 at P_(W), is used as plunger to push down theexpanded solution and to maintain constant pressure within the reactorduring the depressurization stage. The presence of filter FL allowscollecting any precipitate that may have formed during the process. Thevesicles formed are collected in container C and subsequently stored inglass bottles at 4° C. Once depressurization is finished, V-6and V-2 areclosed and depressurization of the equipment proceeds by reopening V-6.

In one embodiment of the invention, the relationship between the amountof CF and organic solvent corresponds to a CF molar fraction ofapproximately 0.3 to 0.95; preferably from 0.5 to 0.8. In one particularembodiment, the dissolution of cholesterol (or derivatives thereof) in aCF is performed in a reactor at a pressure P_(w) of approximately 1 to30 MPa, and a T_(W) of approximately 10 to 70° C. Preferably theapproximate temperature of the reactor is between 10 and 50° C.

In the process of the invention, EGF is dissolved in an aqueous solutioncontaining a cationic surfactant, the concentration of which is aboveits critical micellar concentration.

Surprisingly, the cholesterol:QUATs:EGF vesicles, synthesized using theprocess described above, have yields of EGF vesicle incorporation veryclose to 100%, which are significantly higher than those expected forthe incorporation of any water-soluble molecule, taking into account theresults formerly reported for gentamicin. These yields incholesterol:QUATs:EGF vesicles are also notably higher than thoseobtained for the incorporation of other proteins with structuralproperties similar to EGF, as the water-soluble protein bovine serumalbumin (BSA). This occurs even when the formerly described DELOS-SUSPprocedure is used for said BSA incorporation, as only an incorporationyield of 42% is obtained.

Furthermore, and also in a surprising way, these yields of EGFincorporation into cholesterol:QUATs vesicles are clearly greater thanthose obtained for cholesterol:DPPC:EGF vesicles, even if the DELOS-SUSPprocess is used to prepare them.

Another aspect of the present invention is a pharmaceutical compositioncomprising vesicles comprising EGF, a cationic surfactant andcholesterol or derivatives thereof and at least one pharmaceuticallyacceptable excipient. The pharmaceutically acceptable excipient orexcipients, which form part of the pharmaceutical composition of thepresent invention, can enhance the vesicle activity. Alternatively, theycan help to the handling and processing of the composition of thepresent invention. The EGF vesicles of the present invention may beformulated in several pharmaceutical forms, such as: injectable, spray,gels, viscous solutions, creams, ointments, transdermal patches, depots,inhaled formulations and others known to those skilled in this technicalfield.

Various excipients can be mixed during the synthesis of the vesicles ofthis invention or after it to form a suitable material for the abovementioned dosage forms. Generally, excipients; such as solubilizers orsolvents, surfactants, pH modifiers, antioxidants, diluents, matrixsystems, complexing agents, viscosity enhancers, dispersants,humectants, colorants, flavorings, preservatives, permeability enhancersand others; can be used for usual purposes and in typical quantitieswithout affecting the characteristics of the compositions of the presentinvention, as known by those skilled in this technical field(Remington's Pharmaceutical Sciences (1995)). Additional examples ofexcipients that may be used in pharmaceutical formulations of EGFvesicles can be found in the Handbook of Pharmaceutical Excipients (6thedition).

In an embodiment of the present invention, the pharmaceuticalcomposition is a controlled or sustained release form. Forms ofsustained or controlled release usually include matrix systems, ionexchange resins, or individual barriers to control the diffusion of theEGF vesicles.

In one embodiment of the invention, for the preparation ofpharmaceutical compositions, EGF vesicles are conditioned by a processof concentration-diafiltration, using known apparatus in this technicalbranch. The pharmaceutical compositions of the invention may beadministered by various routes, among them: systemic, intralesional,mucosal, topical, transdermal, ophthalmic, or as inhaled formulation.

In another aspect, the present invention comprises the use of vesiclesof EGF, a cationic surfactant, and cholesterol or derivatives thereof inthe manufacture of a medicament. In an embodiment of the invention, saidmedicament is intended for the treatment of diseases in which isrequired help in the healing and tissue regeneration processes in anymammalian species; wherein cationic surfactants, cholesterol and EGFforming said vesicles are acceptable in pharmaceutics. In a preferredembodiment, the mammal is a human.

Said medical use comprises administering an effective quantity of saidvesicles to treat a disease requiring exogenous EGF administration toregulate the processes of proliferation, growth and migration ofepithelial cells or to induce angiogenesis.

In general, it is considered that an effective concentration for theadministration of EGF vesicles of the present invention (or thecomposition comprising them) would be 1.0 to 200 μg/mL EGF equivalents,preferably 5.0 to 100 μg/mL EGF equivalents per administration. Thevolume and frequency of administration depends on the type of lesion,its size and the administration device used, as is well known by expertsskilled in the art. It may also be appropriate to administer therequired dose in two, three, four or more sub-doses at the appropriateintervals during the day.

The exact dosage and frequency of administration depends on theparticular condition being treated and its severity, age, weight, sex,extent of disease, and the general physical condition of the patient, aswell as on any other concomitant medications administered to theindividual, as it is well known by those skilled in the art. Further, itis evident that the effective daily amount may decrease or increase,depending on how the patient responds to the medication, and/or theassessment made by the doctor who prescribed the drug of the presentinvention. Thus, the daily effective quantities listed above are to beconsidered as guidelines or recommendations.

In one embodiment of the invention, the medicament manufactured with thevesicles of the invention is used for treating complex wounds of anyperipheral soft tissue. In a particular embodiment, the complex wound isa diabetic foot ulcer. In another particular embodiment, the drug isused for the treatment of venous ulcers, decubitus ulcers or burns.

In another embodiment of the invention, the medicament is used to treata disease such as adult respiratory distress syndrome. The medicamentmanufactured with the vesicles of the invention is also useful for thetreatment of digestive tract lesions such as ulcerative colitis,duodenal ulcers and distal colitis. In another embodiment, the drug isused for the treatment of eye lesions.

A cosmetic product characterized by comprising vesicles of EGF, acationic surfactant, and cholesterol or derivatives thereof, and atleast one acceptable excipient for cosmetics or dermal pharmaceuticalsis also part of the present invention. In this aspect of the invention,cationic surfactants, cholesterol (or derivatives thereof) and EGF areacceptable in pharmaceutics and cosmetics.

The EGF vesicles of the present invention can be formulated into variouscosmetic or dermal pharmaceutical forms, as solids, liquids andsemisolids, such as and not restricted to: injections, spray atomizedliquids, gels, creams, multiple emulsions, aqueous dispersions, milks,balsams, lotions, foams, sera, ointments, transdermal patches, wipes,depots, balms, powders, bars, inhaled formulations and the like, in allcases, including the rinse and the permanence formulations.

In general, the cosmetic or dermal pharmaceutical composition of theinvention may contain excipients such as, but not limited to,solubilizers or solvents, surfactants, pH modifiers, antioxidants,diluents, matrix systems, complexing agents, viscosity enhancers,dispersants, humectants, gelling polymers, thickeners, softeners,stabilizers, odor absorbents, chelating agents, plant extracts,essential oils, marine extracts, agents coming from a biofermentationprocess, mineral salts, cell extracts and sunfilters (photoprotectiveagents of organic or mineral nature, active against A and/or Bultraviolet rays), pigments or dyes, flavorings, preservatives,permeability enhancers and others, and mixtures thereof, provided thatthey are physically and chemically compatible with the other componentsof the composition of the present invention. These excipients may beused for the usual purposes and in typical amounts without affecting thecharacteristics of the compositions of the present invention, as knownby those skilled in this technical field (Additional examples can befound described in the CTFA Cosmetic Ingredient Handbook, TwelfthEdition (2008)). The nature of such additional adjuvants may besynthetic or natural in origin, like for example plant extracts, orcoming from a biofermentation process.

Therefore, the use of the EGF vesicles previously described for themanufacture of a cosmetic product is also an object of the presentinvention. In an embodiment of the invention, the cosmetic product isfor preventing senescence and aging of the skin.

EXAMPLES

The following examples are shown for illustrative purposes and shouldnot be considered as limitations to the invention

Example 1 Synthesis of Cholesterol:DPPC:EGF Vesicles Using CompressedFluid Technology

Firstly, a solution of 12 mg of cholesterol and 24 mg of DPPC in 1.2 mLof ethanol is introduced into a high pressure reactor of 6 mL volume, atatmospheric pressure and temperature (Tw=35° C.). Compressed CO₂ isadded, causing volumetric expansion of the solution to reach a molarfraction X_(CO2)=0.7 and a working pressure P_(w)=10 MPa. The system isleft under agitation during approximately 60 minutes at 10 MPa and 35°C., to achieve full homogenization and thermal balance. Finally, theexpanded organic solution is depressurized from the working pressure toatmospheric pressure, on 24 mL of an aqueous solution of EGF at thedesired concentration (between 15 μM and 40 μM). In this last step, astream of N₂ at 10 MPa is used as plunger to push down the volumetricexpanded solution to maintain a constant working pressure in the reactorduring depressurization.

Subsequently, the vesicles are transferred to a hermetically sealedcontainer and stored until use at 5±3° C.

As a result, DPPC:cholesterol (1:1) vesicles were obtained, with EGFincorporated at a concentration between 15 μM and 40 μM. The physicalappearance, mean size, particle size distribution and Z-potential areshown in Table 1. The mean size, particle size distribution andZ-potential were determined by DLS.

It can be seen that the various vesicle preparations have no short-termstability problems, and have relatively small mean size andpolydispersity index (PDI), which makes them attractive from thepharmaceutical viewpoint. However, the absolute Z-potential is verysmall (<+10 mV), well below the values that are considered to allowcolloidal stability of dispersed systems, which are normally absolutevalues above 30 mV (Carrion et al., J. Colloid. Interface Sci. 1994,164: 78-87). This characteristic indicates that the long-term stabilityof this vesicle system may be compromised.

TABLE 1 Physical appearance, mean particle size and Z-potential of thedifferent variants of cholesterol:DPPC:EGF vesicles for differentcompositions. Composition (EGF:choles- Mean size Z-Potential terol)Physical Appearance (nm) (PDI)* (mV) (±SD)*  0 μM:1M Disperse opalescent143.2 (0.180)  +4.8 (±2.43) solution 15 μM:1M Disperse opalescent 188.3(0.180) +3.92 (±2.43) solution 25 μM:1M Disperse opalescent 226.4(0.297) +5.12 (±3.99) solution 40 μM:1M Disperse opalescent 227.3(0.243) +1.78 (±0.82) solution *Nano-ZS (Malvern Instruments, UnitedKingdom), PDI—polydispersity index

Example 2 Synthesis of Cholesterol:CTAB:EGF Vesicles Using CompressedFluid Technology

Firstly, a solution of 76 mg of cholesterol in 2.88 mL of ethanol isintroduced in a high pressure reactor that has a volume of 6 mL, atatmospheric pressure and working temperature (Tw=35° C.). Compressed CO₂is added, producing volumetric expansion of the solution to reach amolar fraction X_(CO2)=0.7 and a working pressure P_(w)=10 MPa. Toachieve full homogenization and thermal balance, the system is allowedto rest for about 60 minutes at 10 MPa and 35° C. Finally, the expandedorganic solution is depressurized from the working pressure toatmospheric pressure, on 24 mL of a solution of CTAB in mQ water (C=2.83mg/mL) containing EGF at the desired concentration (between 1 and 40μM). In this last step, a stream of N₂ at 10 MPa is used as plunger topush the cholesterol in an ethanol solution to maintain a constantworking pressure in the reactor during depressurization. Then, thevesicles are transferred to a hermetically sealed container and storeduntil use at 5±3° C.

The morphology of the vesicles was evaluated by cryo-TEM, according to aprocedure that has been previously described (Progress in MolecularBiology and Translational Science, Elsevier, 2011, vol. 104, pp. 1-52).

CTAB:cholesterol (1:1) vesicles, with EGF incorporated at aconcentration between 1 μM and 40 μM, were obtained. The results forphysical appearance, mean size and Z-potential are shown in Table 2; theparticle size distribution, in FIG. 2A and the size and morphology, bycryo-TEM, in FIG. 3A. As it can be seen in Table 2, the differentpreparations, with varying proportions of EGF:cholesterol, were stable.The Z-potentials of all the preparations were positive, and far above+30 mV, which predicts elevated long-term stability. An increase in themean size and PDI with an increase in the proportion of EGF:cholesterolcan also be noticed. In FIG. 2A, very highly homogenous particle sizedistributions are observed, having an average diameter not exceeding the200 nm. FIG. 3A shows that, in relation to vesicle morphology studied bycryo-TEM, spheroidal shapes with unilamellar structure predominate. Thementioned characteristics of the different vesicle preparations makethem very attractive from the pharmaceutical viewpoint.

TABLE 2 Physical appearance, mean particle size and Z- potential of thedifferent variants of cholesterol:CTAB:EGF vesicles for differentcompositions Composition (EGF:choles- Mean size Z-Potencial terol)Physical appearance (nm) (PDI)* (mV) (±SD)* 0 μM:1M Disperse opalescent117.7 (0.244)  +73.1 (±11.0) solution 1 μM:1M Disperse opalescent 113.6(0.299)  +75.3 (±10.6) solution 2 μM:1M Disperse opalescent 113.7(0.274) +72.1 (±5.2) solution 5 μM:1M Disperse opalescent  96.6 (0.257)+70.9 (±9.6) solution 15 μM:1M  Disperse opalescent 128.4 (0.379)  +74.2(±10.3) solution 25 μM:1M  Disperse opalescent 145.4 (0.391) +74.8(±6.3) solution 40 μM:1M  Disperse opalescent 180.6 (0.450) +68.40(±5.8)  solution *Nano-ZS (Malvern Instruments, United Kingdom),PDI—polydispersity index

Example 3 Synthesis of Cholesterol:Cetrimide:EGF Vesicles UsingCompressed Fluid Technology

These vesicles were synthesized in a similar way to those in Example 2,but in this case, an aqueous solution of cetrimide with a concentrationof 2.61 mg/mL was used. Cetrimide:cholesterol (1:1) vesicles wereobtained, with EGF incorporated at a concentration between 1 μM and 40μM. The results of physical appearance, mean size and Z-potential areshown in Table 3; the particle size distribution, can be observed inFIG. 2B, and the size and morphology, by cryo-TEM, are shown in FIG. 3B.As it can be seen in Table 3, all the preparations were stable withsmall mean size and PDI values. The mean size, particle sizedistribution and the Z-potential were determined by DLS. The Z-potentialof all the preparations is positive, and well above +30 mV, whichpredicts high long-term stability. FIG. 2B shows that the distributionsof particle sizes are highly uniform, and their average diameter doesnot exceed 200 nm. FIG. 3B shows that spheroidal shapes with unilamellarstructure predominate, according to cryo-TEM. The different vesiclepreparations also have characteristics which make them very attractivefrom the pharmaceutical viewpoint.

TABLE 3 Physical appearance, mean particle size and Z-potential ofdifferent of cholesterol:cetrimide:EGF vesicles for differentcompositions Composition (EGF:choles- Mean size Z-Potencial terol)Physical appearance (nm) (PDI)* (mV) (±SD)* 0 μM:1M Disperse opalescent118.6 (0.392) +78.1 (±16.1) solution 1 μM:1M Disperse opalescent 123.8(0.381) +75.4 (±12.6) solution 2 μM:1M Disperse opalescent 127.3 (0.376)+77.3 (±9.3)  solution 5 μM:1M Disperse opalescent 140.8 (0.375) +79.1(±13.1) solution 15 μM:1M  Disperse opalescent 124.0 (0.388) +71.1(±11.6) solution 25 μM:1M  Disperse opalescent 125.1 (0.436) +70.4(±10.8) solution 40 μM:1M  Disperse opalescent 152.1 (0.44)  +72.7(±11.2) solution *Nano-ZS (Malvern Instruments, United Kingdom),PDI—polydispersity index

Example 4 Synthesis of Cholesterol:BKC:EGF Vesicles Using CompressedFluid Technology

These vesicles were synthesized in a similar way to those in Example 2,but in this example, on one hand, it was used a solution of 81.46 mg ofcholesterol in 2.88 mL of ethanol, and on the other one, an aqueoussolution of 3.0 mg/mL of BKC and EGF at the desired concentration wasused. BKC:cholesterol (1:1) vesicles were obtained, with EGFincorporated at a concentration of 5 μM. The results for physicalappearance, mean size and Z-potential are shown in Table 4. It can beobserved that the vesicle preparation is stable and it has a relativelysmall mean size and PDI. The mean size, particle size distribution andthe Z-potential were determined by DLS. Furthermore, similar to theabove mentioned vesicle preparations, that comprise in their compositiona cationic surfactant of quaternary ammonium type, they have positiveZ-potential values much higher than +30 mV, which predicts highlong-term stability for them. FIG. 3C shows that spheroidal shapes withunilamellar structure predominate, according to the vesicle morphologystudy performed by cryo-TEM. These vesicles also have features that makethem very attractive from a pharmaceutical point of view.

TABLE 4 Physical appearance, mean particle size and Z-potential of thecholesterol:BKC:EGF vesicles at the working composition. Composition(EGF:choles- Mean size Z-Potencial terol) Physical appearance (nm)(PDI)* (mV) (±SD)* 5 μM:1M Disperse opalescent 199.9 (0.335) +74.4(±10.0) solution *Nano-ZS (Malvern Instruments, United Kingdom),PDI—polydispersity index

Example 5 Synthesis of Cholesterol:CTAB:BSA Vesicles Using CompressedFluid Technology

These vesicles were synthesized in a similar way to those in Example 2,but in this case the protein BSA was used in an aqueous solution.CTAB:cholesterol (1:1) vesicles were obtained, with BSA incorporated ata concentration of 0.37 μM, corresponding to 25 μg/mL. The results forphysical appearance, mean size and Z-potential are shown in Table 5. Itcan be seen that the vesicle preparation is stable and they haverelatively small mean size and PDI. The mean size, particle sizedistribution and the Z-potential were determined by DLS. They alsoexhibit positive Z-potential values, well above +30 mV, which predictshigh long-term stability.

TABLE 5 Physical appearance, mean particle size and Z-potential ofcholesterol:CTAB:BSA vesicles at the working composition Composition(BSA:choles- Mean size Z-Potencial terol) Physical appearance (nm)(PDI)* (mV) (±SD)*   0 μM:1M Disperse opalescent 124.2 (0.283) +70.1(±11.0) solution 0.37 μM:1M Disperse opalescent 119.9 (0.378) +76.5(±10.6) solution *Nano-ZS (Malvern Instruments, United Kingdom),PDI—polydispersity index

Example 6 Determination of the Efficiency of Protein Incorporation inthe Vesicles

To determine the efficiency of EGF incorporation in the vesicles, theultracentrifugation method was used for separating free EGF vesicles.From the different vesicle suspensions evaluated, 1.0 mL was taken;placed in vials and centrifuged at high speed (100,000×g) for 60 min at4° C. Subsequently, the protein content in the supernatant (free EGF)was determined by a solid-phase immunoenzymatic assay (ELISA) (Vázquezet al., Biotecnol. Apl. 1990, 7: 42-49). The efficiency of EGFincorporation in the vesicles was determined by the followingexpression:

${{Efficiency}\mspace{14mu}{of}\mspace{14mu}{incorporation}\mspace{14mu}(\%)} = \frac{\left\lbrack {{E\; G\; F\mspace{14mu}{initial}\mspace{14mu}{mass}} - {E\; G\; F\mspace{14mu}{mass}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{supernatant}}} \right\rbrack*100}{E\; G\; F\mspace{14mu}{initial}\mspace{14mu}{mass}}$

The results are shown in Table 6. Results of the efficiency of EGFincorporation in vesicles with the DPPC:cholesterol composition(obtained in Example 1), the CTAB:cholesterol composition (obtained inExample 2) and the cetrimide:cholesterol composition (obtained inExample 3) are shown. The effect of the EGF concentration(EGF:cholesterol ratio) were also evaluated. Values of highEGF-incorporation efficiency (near 100%) can be observed inQUATs:cholesterol systems, for a wide range of EGF concentrations(EGF:cholesterol ratio). However, the results of the efficiency of EGFincorporation into the DPPC:cholesterol system were very low, since themaximum efficiency of incorporation did not exceed 10%, for the highestEGF concentration.

TABLE 6 Efficiency of EGF incorporation into vesicles of differentcompositions Efficiency of incorporation (%) (Mean ± STDEV, n = 2)Composition DPPC- CTAB- cetrimide- (EGF:cholesterol) cholesterolcholesterol cholesterol  1 μM:1M ND 99.8 ± 1.3 99.9 ± 0.5  2 μM:1M ND99.7 ± 0.9 99.3 ± 0.7  5 μM:1M ND 99.7 ± 0.7 99.0 ± 0.3 15 μM:1M 1.0 ±0.2 99.9 ± 0.5 98.5 ± 2.6 25 μM:1M 2.7 ± 0.3 99.7 ± 0.2 98.0 ± 0.5 40μM:1M 8.7 ± 0.6 99.9 ± 0.4 97.9 ± 1.8 ND—Not determined

In the particular case of the vesicles comprising BSA, the sameprocedure described for EGF was followed, except that the quantificationof the protein was performed by the bicinchoninic acid method(Micro-BCA) (Smith et al., Anal. Biochem. 1985, 150: 76-85). Theefficiency of BSA incorporation in the CTAB:cholesterol vesicles,prepared as described in Example 5, was only 42±5%. When the efficiencyof BSA incorporation was compared with the EGF incorporation (shown inTable 6), in vesicles with the same CTAB:cholesterol composition, it canbe observed that EGF incorporation was significantly higher.

Example 7 Reproducibility of the Synthesis of Cholesterol:CTAB:EGFVesicles by Compressed Fluid Technology

Aiming to test the robustness and reproducibility of the methodologyused for the synthesis of EGF vesicles, the mean particle size, PDI,Z-potential and efficiency of EGF incorporation were determined inseveral batches of 5 μM:1 M and 15 μM:1 M composition ofEGF:cholesterol, prepared on different dates. In Tables 7 and 8, theresults obtained are shown. The mean particle size, PDI and Z-potentialwere determined by DLS. The efficiency of EGF incorporation wasdetermined as described in Example 6.

TABLE 7 Features of CTAB:cholesterol (1:1) vesicles with EGFincorporated at a concentration of 5 μM, after preparation by DELOS-SUSPParticle EGF incor- Code diameter Z-Potential poration (Date) (nm)* PDI(mV)* (%) Batch 1 103.3 0.363 69.5 98.1 (26 Oct. 2010) Batch 2 96.750.376 79.7 97.3 (29 Oct. 2010) Batch 4 101.4 0.311 77 97.2 (10 Feb.2011) Batch 8 92.82 0.351 76 96.6 (18 Nov. 2011) Batch 10 113.6 0.28175.9 95.8 (10 Feb. 2012) Batch 11 111.3 0.335 70.1 98.6 (10 Feb. 2012)*Nano-ZS (Malvern Instruments, United Kingdom), PDI—polydispersity index

TABLE 8 Characteristics of CTAB:cholesterol (1:1) vesicles with EGFincorporated at a concentration of 15 μM after preparation by DELOS-SUSPParticle EGF incor- Code diameter Z-Potencial poration (Date) (nm)* PDI(mV)* (%) Batch 3 141.7 0.419 80.4 99.4 (29 Oct. 2010) Batch 5 139.50.278 74 98.1 (24 Feb. 2011) Batch 6 142.2 0.275 72.9 99.7 (28 Feb.2011) Batch 7 139 0.353 77 99.1 (29 Jun. 2011) Batch 9 140.9 0.357 77.598.6 (30 Jun. 2011) Batch 8 141.4 0.319 76.0 99.5 12 Jul. 2011 *Nano-ZS(Malvern Instruments, United Kingdom), PDI—polydispersity index

From the values reported in Tables 7 and 8, it can be concluded that thevesicles have similar characteristics, independently of the date ofpreparation, which allows to state that the DELOS-SUSP method isreproducible and robust for preparation of the EGF vesicles.

Example 8 Scale Up of the Synthesis of Cholesterol:CTAB:EGF VesiclesUsing Compressed Fluid Technology

The vesicles were synthesized as in Example 2, but on a 50 times greaterscale. First, a solution of 3.8 g of cholesterol in 144 mL of ethanol isintroduced in the high pressure reactor of 300 mL volume at atmosphericpressure and working temperature (Tw=35° C.). Compressed CO₂ is addedproducing volumetric expansion of the solution to reach a molar fractionX_(CO2)=0.7 and a working pressure P_(w)=10 MPa. To achieve fullhomogenization and thermal balance, the system is left for about 60minutes at 10 MPa and 35° C. Finally, the expanded liquid solution isdepressurized from the working pressure to atmospheric pressure, on 1200mL of a solution of CTAB in mQ water (C=2.83 mg/mL) containing EGF atthe desired concentration (5 and 12 μM). In this last step, a stream ofN₂ at 10 MPa is used as a plunger to push the cholesterol in ethanolsolution to maintain constant the working pressure inside the reactorduring depressurization. Then the vesicles are transferred to ahermetically sealed container and stored until use at 5±3° C.CTAB:cholesterol (1:1) vesicles with EGF incorporated at a concentrationof 5 μM and 12 μM were obtained. The results of physical appearance,mean size, PDI and Z-potential are shown in Table 9. The mean particlesize, PDI and Z-potential were determined by DLS.

TABLE 9 Physical appearance, mean particle size, PDI and Z-potential ofthe variants of cholesterol:CTAB:EGF vesicles, for differentcompositions, after preparation by DELOS-SUSP, at a pilot scaleComposition (EGF:choles- Mean size Z-Potencial terol) Physicalappearance (nm) (PDI)* (mV) (±SD)* 0 μM:1M Disperse opalescent 132.7(0.224) +63.1 (±2.0) solution 5 μM:1M Disperse opalescent  127 (0.211)+61.3 (±2.2) solution 12 μM:1M  Disperse opalescent 160.7 (0.234) +60.1(±2.6) solution *Nano-ZS (Malvern Instruments, United Kingdom),PDI—polydispersity index

Table 9 shows that EGF:CTAB:cholesterol vesicles are stable atproportions of 5 μM:1 M and 12 μM:1 M. It can be seen that in the scaleup process the particles obtained have physico-chemical characteristicscomparable to those obtained in the 6 mL scale (Example 2). In bothcases, the mean sizes of the particles obtained are under 200 nm.

Example 9 Biological Activity of the Vesicles, Evaluated by the CellProliferation Assay in Murine A31 3T3 Fibroblasts

The biological activity of the EGF vesicles, prepared as described inExamples 1 to 3, was determined using a cell proliferation assay(Mire-Sluis and Page, J. Immunol. Methods 1995, 187: 191-199). In thiscase, the ability of free EGF, EGF liposomes and the different vesiclescomprising EGF to increase cell proliferation of the A31 3T3 line ofmurine fibroblasts was evaluated. The biological activity of thedifferent vesicular suspensions was evaluated by applying an appropriatedilution of the suspension directly onto the test cells, in the way thatthe absorbance of the samples fell within the range of the curve of theworking reference material, which was previously calibrated against theinternational reference material EGF 91/550, provided by the NationalInstitute for Biological Standards and Control (NIBSC, United Kingdom).

With the objective of comparing the potency of the various EGF vesiclepreparations with free EGF, the specific activity of the differentpreparations was calculated from the biological activity results, by thefollowing expression:

${E\; G\; F\mspace{14mu}{specific}\mspace{14mu}{activity}\mspace{14mu}\left( {{IU}\text{/}{mg}} \right)} = \frac{E\; G\; F\mspace{14mu}{biological}\mspace{14mu}{activity}\mspace{14mu}\left( {{IU}\text{/}{mL}} \right)}{E\; G\; F\mspace{14mu}{concentration}\mspace{14mu}\left( {{mg}\text{/}{mL}} \right)}$

The biological activity is measured by the assay described and theprotein concentration is given by the nominal value (in mg/mL) of theequivalent EGF concentration in the different vesicle preparations.

As a control in the cell proliferation assay, blank vesicles (withoutthe addition of EGF) were used, in correspondence with the differentvariants tested. In these samples, at dilutions lower than those usedfor the vesicles with EGF, neither a cytotoxic effect nor an increase inthe proliferation was detected.

FIG. 4A shows that EGF vesicles with CTAB:cholesterol compositionexhibit increased specific activity as compared to free EGF and EGF invesicles with the DPPC:cholesterol composition. In FIG. 4B, a resultsimilar to the former is also observed when cetrimide, instead of CTAB,was used in the composition of the vesicles prepared by the sameprocedure. The purpose of this evaluation was to determine if the EGFbiological function is affected by the components or by the synthesisprocess of said vesicles. However, for the vesicles withQUATs:cholesterol composition, an unexpected increase in the specificbiological activity of EGF was found.

Example 10 Protease Resistance of EGF Integrated into QUATs:CholesterolVesicles

EGF vesicles with QUATs:colesterol (1:1) compositions, prepared as inExamples 2 and 3 of the present invention, were used. This experimentwas performed to evaluate the ability of QUATs:cholesterol vesicles topreserve the stability of integrated EGF against proteases. It is knownthat chronic wounds, such as diabetic foot ulcers, have proteolyticenvironments that may affect the bioavailability of the drugs used totreat them (Bennett and Schultz, Am. J. Surg. 1993, 166: 74-81).

For this evaluation, trypsin was used as a model protease. The enzymaticreaction was prepared in Tris-HCl buffer with pH 8.5 and 20 mMconcentration, containing a final concentration of 0.5 μg/mL of trypsin.The final concentration of free EGF or the EGF equivalent in thedifferent vesicle preparations was 125 μg/mL. Incubation of the sampleswas performed for periods of 4, 8, 16 or 24 hours at 37° C.Trifluoroacetic acid (TFA), at a final concentration of 0.1% (v/v) wasused to stop the enzymatic reaction.

After stopping the reaction, samples were diluted in absolute methanol,to a final methanol concentration of 80% (v/v), stirred and centrifugedin a table centrifuge at 10 000 rpm for 5 minutes. Finally, thecentrifugation supernatants were filtered through polycarbonate filtersof 0.2 μm pore size and then applied to a HPLC system (Merck, Germany).The EGF standard and vesicle samples were analyzed using a C18 reversephase column (Vydac, Hesperia, Calif., USA) and detected at 226 nm. Todo this, a linear gradient of 20% to 40% of B for 28 minutes was used.Mobile phase A consists of 0.1% TFA/water and mobile phase B consists of0.05% TFA/Acetonitrile. The injection volume analyzed was 5.0 mL,corresponding to about 20 μg of EGF. The flow rate used was 1.0mL/minute. The EGF concentration in the samples was quantified byinterpolation using a calibration curve of the EGF in the area under themain peak and the EGF concentrations of known samples, from thechromatograms obtained at 226 nm. The percentage of EGF remaining ineach sample, after incubation with trypsin, was calculated using thefollowing expression:

${{Remaining}\mspace{14mu} E\; G\; F\mspace{14mu}(\%)} = \frac{E\; G\; F\mspace{14mu}{{conc}.\mspace{14mu}{after}}\mspace{14mu}{trypsin}\mspace{14mu}{incubation}\mspace{14mu}\left( {{µg}\text{/}{mL}} \right)*100}{E\; G\; F\mspace{14mu}{{conc}.\mspace{14mu}{before}}\mspace{14mu}{trypsin}\mspace{14mu}{incubation}\mspace{14mu}\left( {{µg}\text{/}{mL}} \right)}$

FIG. 5A shows that EGF vesicles with CTAB:cholesterol compositionexhibit increased stability against trypsin, compared to free EGF,during a period of 24 hours. Similarly, FIG. 5B, shows that EGF vesicleswith cetrimide:cholesterol composition show a similar behavior to theone described before. Significant differences were not found among thedifferent EGF loads evaluated (EGF:cholesterol ratio).

The results of the stability to proteases, of EGF incorporated inQUATs:cholesterol vesicles, showed that EGF vesicles have much higherstability than free EGF.

Example 11 Demonstration of the Antimicrobial Activity ofQUATs:Cholesterol Vesicles

Vesicle suspensions comprising QUATs:cholesterol were evaluated todetermine if they showed antimicrobial activity. This activity wasdetermined using the agar diffusion method (Manual of ClinicalMicrobiology. 6th ed. Washington, D.C.: ASM; 1995). The effectiveness ofthe different suspensions was assayed against Gram positive bacteria(Bacillus subtilis, Staphylococcus aureus), Gram negative bacteria (E.coli, Proteus mirabilis), and against fungi (Candida albicans,Aspergillus niger) using the technique of wells in nutrient agar plates.These microorganisms were identified and provided by the microorganismcollection BCCM/LMG (Belgium). Bacteria were grown overnight at 37° C.in Tryptone Soy Broth (Oxoid), and fungi were incubated for 72 hours at28° C. in Sabouraud Cextrose Broth (Oxoid). These suspensions were usedas inocula. A final inoculum, using 100 μl of a suspension containing10⁸ colony forming units/ml of bacteria, or 10⁴ spores/ml of fungus, wasspread on Tryptone Soy Agar and Sabouraud Dextrose Agar (Oxoid) plates,respectively. The disk (6 mm in diameter) was impregnated with each ofthe different vesicle suspensions to be tested. Ciprofloxacin andfluconazole (100 μg/ml) were used as positive controls for bacteria andfungi, respectively. Assay plates were incubated at 37° C. for 24 hoursfor bacteria, and 28° C. during 72 hours for fungi, depending on theincubation time required for visible growth.

Table 10 shows that the different vesicle suspensions had antimicrobialeffect against Gram positive bacteria and fungi. In general,microorganisms were more sensitive to vesicular suspensions ofcetrimide:cholesterol composition than those of CTAB:cholesterol. Somevesicular suspensions showed antibacterial and antifungal activityagainst certain microorganisms comparable to ciprofloxacin andfluconazole, respectively.

TABLE 10 Antimicrobial and antifungal activity of different vesiclesuspensions of QUATs:cholesterol against bacteria and fungi.Antimicrobial activity Fungicidal activity (Inhibition zone, mm)(Inhibition zone, mm) Vesicular suspensions B. subtilis S. aureus E.coli P. mirabilis A. niger C. albicans CTAB:cholesterol (1:1) 22 18 R 1016 16 CTAB:cholesterol (1:1) with 12  6 R  4  6  6 EGF incorporated at 5μM Cetrimide:cholesterol (1:1) 26 16 4 14 18 18 Cetrimide:cholesterol(1:1) with 24 18 4 10 18 18 EGF incorporated at 5 μM Control — — — — — —Ciprofloxacin 22 20 18  18 — — Fluconazole — — — — 21 22 R—Resistant

Example 12 Manufacture of a Liquid Spray Atomizer Formulation ContainingEGF Vesicles

A vesicle suspension of CTAB:cholesterol (1:1) with EGF:cholesterolcomposition of 5 μM: 1 M, obtained as in Example 2, with EGF equivalentconcentration of 25 μg/mL, was diluted to the EGF equivalentconcentration of 15 μg/mL in 10 mM sodium phosphate buffer pH 7.2. Thisfinal solution also contains 17% (v/v) glycerol, 10% (v/v) ethanol,0.02% (w/v) butyl hydroxytoluene (BHT), 0.18% (w/v) of methyl parabenand 0.02% (w/v) of propyl paraben. The resulting dispersion was filteredthrough 0.2 μm cellulose acetate sterilizing filters, and dispensed inglass vials under nitrogen atmosphere.

Example 13 Manufacture of a Gel Formulation Containing EGF Vesicles

A vesicle suspension of cetrimide:cholesterol (1:1) with EGF:cholesterolcomposition 5 μM: 1 M, obtained as in Example 3, with EGF equivalentconcentration 25 μg/mL, was diluted to the equivalent concentration of15 μg/mL This formulation contains 10 mM Tris-HCl buffer pH 7.2, andCarbomer (Carbopol 940), to a final concentration of 1.25% (w/v);glycerol, 3% (w/v) and 20 mM of L-methionine. Furthermore, theformulation contains 0.02% (w/v) BHT, and as antimicrobial preservative0.18% (w/v) methyl paraben and 0.02% (w/v) propyl paraben.

Example 14 Manufacture of a Parenteral Formulation Containing EGFVesicles

A vesicle suspension of BKC:cholesterol (1:1), with EGF:cholesterol 5μM:1 M composition, obtained as in Example 4, with EGF equivalentconcentration of 25 μg/mL, was introduced in the cell of the tangentialflow ultrafiltration device Sartocon Slice 200. For ultrafiltration ofthe vesicles, cassettes with Hydrosart® membranes of 30 kDa pore sizewere used. The concentration-diafiltration process is conducted at atemperature of 25±3° C. and the maximum pressure drop at the entrance ofthe cassette was maintained lower than 4 bar. During the process, thesuspension was concentrated 5 times (final equivalent concentration ofEGF of 125 μg/mL). After the concentration step, the vesicles werediluted in 10 mM sodium phosphate buffer pH 7.2, to an equivalentconcentration of 75 μg/mL of EGF. This formulation also contains 20 mMof L-methionine and 0.02% (w/v) of BHT. The resulting suspension wasfiltered through a 0.2 μm sterilizing filter and dispensed in glassvials under nitrogen atmosphere.

Example 15 Comparison of the Pharmacodynamic Effect of Liposomes andVesicles Comprising EGF in Animal Models of Wound Healing

Experimental Methodology

To evaluate the pharmacological efficacy of the formulations listedbelow, an experimental model of chronic ulcer of total thickness wasdeveloped on the back of rats. Sprague Dawley rats of 250-270 gramsweight, which were randomly assigned to form 7 experimental groups, of10 animals each, were used. Rats were intraperitoneally anesthetizedwith a combination of ketamine/xylazine to extensively depilate theirdorsal region. Two symmetrical bilateral retroscapular ulcers were madeof 8 mm diameter and total thickness up to the upper fascia, which wasspared. Immediate application of triamcinolone acetonide, as compresses,was begun, once a day during the first three days, to stop healing andinduce the characteristic changes of chronicity. After 7 days, theinterruption of the healing process and chronicity of the ulcers werecorroborated by the absence of granulation tissue and hypertrophy of theepithelial edges. From this moment, application of the studiedtreatments started as described below:

-   Group 1: treatment-free (saline). They were subjected to the same    conditions of handling and manipulation of the rest of the groups.    Sterile physiological saline solution was applied in nebulized form.-   Group 2: empty DPPC-cholesterol liposomes,-   Group 3: empty CTAB-cholesterol vesicles,-   Group 4: empty cetrimide cholesterol vesicles,-   Group 5: DPPC-cholesterol liposomes loaded with EGF at a    concentration of 25 μg/ml,-   Group 6. CTAB-cholesterol vesicles loaded with EGF at 25 μg/ml,-   Group 7. Cetrimide-cholesterol vesicles loaded with EGF at 25 μg/ml.

Wounds were cleaned daily. After sanitizing them, each group wasadministered the suspension indicated in each case. The application ofthe suspensions was performed twice daily, for 14 days. The experimentalsystems were treated by topical application of the suspensions ofvesicles, or liposomes, by nebulization. All animals were subjected toautopsy and sampling 14 days after the onset of the treatment assignedto each group. Samples were fixed in 10% neutral formalin, and 72 hourslater were hemisected uniformly, for later inclusion in paraffin. Thestains used were: hematoxylin-eosin, Mallory's trichrome reaction,Verhoeff's and Gomori's reticulum method. The samples were blindlyanalyzed by two independent researchers.

Results:

It was not necessary to exclude contaminated ulcers; thus, 20 lesionswere used for each of the 6 experimental groups. Briefly, it wascorroborated that all pharmaceutical presentations of the EGF vesiclessignificantly stimulated the overall healing process, when compared togroups with EGF-free vesicles (blank), liposomes loaded with EGF at 25μg/ml, and the experimental control group treated with saline. The datareported relate to the average of two independent studies made atdifferent times. Mann-Whitney U and Student's t-test were used to makethe comparisons. All the parameters met the criteria of normaldistribution. The parameters studied and the results are shown in Table11.

TABLE 11 Pharmacodynamic effect of the suspensions evaluated in achronic wound model in rats Inflammatory Fibroangiogenic Epithelial % ofExperimental infiltrate response migration contraction group (grades1-5) (1-5 points) (en microns) of the edges Group 1: saline 4.61 ± 1.03 2.87 ± 0.65 3.0 ± 1.1 × 10³  23.6 ± 5.11 Group 2: empty 4.27 ± 0.02  3.2 ± 0.96 2.5 ± 0.7 × 10³  18.55 ± 3.21  DPPC-cholesterol liposomesGroup 3: empty 3.8 ± 1.00  2.56 ± 0.93 2.97 ± 1.6 × 10³   21.9 ± 4.63CTAB-cholesterol vesicles Group 4: empty 4.51 ± 0.95  2.64 ± 0.55 2.8 ±1.5 × 10³  31.02 ± 1.81  cetrimide-cholesterol vesicles Group 5: DPPC-2.9 ± 0.88*  3.81 ± 0.87* 4.6 ± 1.04 × 10³*  63.7 ± 6.75* cholesterolliposomes loaded with EGF Group 6: CTAB- 1.18 ± 0.04**   4.2 ± 0.18**6.27 ± 0.27 × 10³**  88.7 ± 6.26** cholesterol vesicles loaded with EGFGroup 7: cetrimide- 1.12 ± 0.07**  4.17 ± 0.25** 7.02 ± 0.53 × 10³** 85.3 ± 7.01** cholesterol vesicles loaded with EGF *Denotes a value ofp ≦ 0.05; **Denotes a value of p ≦ 0.01.

The experiment shows that CTAB-cholesterol and cetrimide-cholesterolvesicles loaded with EGF had a potent healing effect. Theanti-inflammatory effect observed for groups 6 and 7 is notable comparedwith the other treatments. It is possible that the effect of promotingfibroangiogenesis and contraction is connected to the reduction ofbalance of immuno-inflammatory cells infiltrating the neodermis.Similarly, a marked effect promoting epithelial migration was evidenced,microscopically demonstrating the presence of a stratified epidermis.The response obtained for the group treated with EGF-loaded liposomeswas less than the one detected in groups 6 and 7. Superiority of thetreatments including suspensions of vesicles loaded with EGF, applied togroups 6 and 7, was also revealed in a similar experiment wherechronicity in the wounds was induced by topical application ofmethylglyoxal.

Example 16 Treatments Based on the Topical Application of VesiclesComprising EGF in Patients with Diabetic Foot Ulcers

In clinical cases, most of the treated lesions exceeded 90% probabilityof requiring amputation, according to the University of Texas scale.Treatments were administered topically.

General Characteristics of the Treated Patients:

They had diabetes mellitus types I or II of long evolution and weremedicated with insulin, sulfonylureas, or biguanides, as oralhypoglycemic agents. They had a personal history of poor scarring, whilesome of them had experienced prior contralateral amputation. All treatedlower limb injuries corresponded to ischemic or neuropathic diabeticfoot. Lesions, as a whole, were classified as chronic, complex andrecalcitrant. The evolution period of the lesions ranged between lessthan one month and seven years. The size of the treated lesions rangedbetween 20 and 80 square centimeters. The depth in some lesions eveninvolved the periosteum. From an anatomical point of view, the treatedlesions were in the lateral, calcaneus and/or metatarsal regions. Allpatients received the first treatments hospitalized, as long as exeresisprocedures under anesthesia, as well as parenteral antibiotic therapywere necessary. Upon healing, or total wound granulation with evidenceof epithelial migration, the patients were subjected to an outpatientregimen and a follow up on alternate days for cure and medication. Aftercomplete epithelialization, each patient was followed up for 12 monthsafter healing. Evaluation of the occurrence of relapses, registry ofadverse reactions, and assessment of the patient general status werecarried out for this purpose.

Table 12 shows the demographic and epidemiological characteristics ofthe cohort of patients receiving topical treatments.

TABLE 12 Demographic and epidemiological features of the cohort ofpatients that received topical treatment Type Duration Evolution of ofDM Classification of the Identif. Age Sex DM* evolution of the lesionlesion Type of lesion JLG  56 y** M II 15 y Neuropathic 2 years Frontalextended, with ischemic residual after component transmetatarsalamputation OFS 65 y F II 17 y Neuropathic 21 days Wagner III. withproximal Anterolateral part calcified of the foot. Lower pattern rightextremity AVL 52 y M II  9 y Pure 7 years Plantar, neuropathicgranulated for 7 years without evidence of contraction/epithelialization PAT 47 y M II 12 y Ischemic with 32 days Wagner IV.Ankle brachial Transmetatarsal index (ABI) of amputation 0.532 residualbase GMA 69 y M II 10 y Pure 6 months Frontal, residual neuropathicafter transmetatarsal amputation *DM: Diabetes mellitus, **y: years

The treatment followed with each of the patients is described below:

Patient JLG. Male, 56 years old, controlled with insulin and without anyother clinically manifest diabetes complications. Has extensive frontallesion with ischemic component, which persists two years aftertransmetatarsal amputation. There were no granulation andepithelialization processes after establishment of antimicrobial andozone therapies for two years. After performing surgical cleaning andstimulation of the edges, treatment was begun on alternate days usingthe topical spray formulation described in Example 12 containingEGF:CTAB:cholesterol vesicles. In eight weeks of treatment, completeepithelization of the lesion was achieved. The results are shown in FIG.6.

Patient OFS. Female, smoker, obese, with history of blood hypertension,controlled with oral hypoglycemic agents. Rapidly advancing necrosis ofsoft tissue and tendons develops from a local insect bite. Surgicalexeresis of the necrotic material is performed. Antimicrobial therapy isstarted and the lesion is cured on alternate days. Because 14 days afterthe intervention, the granulation process is torpid and slow, treatmentapplication is begun on alternate days using the topical sprayformulation described in Example 12, containing EGF:CTAB:cholesterolvesicles. In eight weeks of treatment, full epithelialization of thelesion was achieved.

Patient AVL. Male, 52 years old, controlled with glibenclamide, Charcotneuropathic plantar foot lesion that has granulated for 7 years, withoutevidence of contraction/epithelialization. After surgical cleaning andedge stimulation, treatment was started on alternate days using thetopical formulation in gel form described in Example 13, containingEGF:cetrimide:cholesterol vesicles. In eight weeks of treatment,complete epithelialization of the lesion was achieved.

Patient PAT. Male, 47 years old, without other co-morbidities orclinically manifest complications of diabetes, even after peripheralarterial disease was detected. As a result of local trauma, atransmetatarsal abscess developed that lead to liquefactive necrosis ofnearly all the forefoot with notable phlogistic signs. Transmetatarsalamputation was performed and antibiotic therapy was begun. As a resultof the amputation residual base unsatisfactory progress after 30 days,having to carry out extensive debridements due to the presence ofischemic microplaques; intervention was started with the topicalformulation in gel form described in Example 13, containingEGF:cetrimide:cholesterol vesicles. The formulation was applied on theedges and wound surface. The treatments were performed on alternate daysand extended during 4 weeks. Since the first administration, thepresence of ischemic plaques was eliminated and a productive andbleeding granulation tissue began to develop, which later facilitatedapplication of a partial-thickness skin-free graft.

Patient GMA. Male, 69 years old, controlled with insulin and withoutclinically manifest diabetes complications. The patient has a frontalresidual lesion that persists six months after transmetatarsalamputation. There were no granulation and epithelialization processesafter 3 months of antimicrobial and ozone therapies. After performingsurgical cleaning, treatment was carried out on alternate days with thetopical spray formulation described in Example 12, containingEGF:CTAB:cholesterol vesicles. In six weeks of treatment, completeepithelialization of the lesion was achieved.

Example 17 Treatments Based on the Infiltration of EGF Vesicles inPatients with Diabetic Foot Ulcers

In clinical cases, most of the treated lesions exceeded 90% probabilityof requiring amputation according to the University of Texas scale.Treatments were administered by infiltration. The general features ofthe treated patients correspond with those described in Example 16.

The parenteral formulation referred to in Example 14, which usesEGF:BKC:cholesterol vesicles was used for the infiltrative treatment.The essence of this treatment consists in intra- and perilesionalinternal injection in the edges and bottom of the lesion, at equidistantpoints. The material is deposited directing the needle to the base ofthe ulcer or the depth of the wedge at an angle of 15-45 degrees, alwaysincluding the dermal-epidermal junction to stimulate contraction of theedges. At each point between 100 and 1000 μL were deposited, dependingon the clinical appearance of the tissue and its characteristics. Thisis performed two to three times per week. The lesions treated anddemographic features of the treated population are described in Table13. In all treated cases, minor or major amputations were prevented.

TABLE 13 Demographic and epidemiological features of the cohort ofpatients that received infiltrative treatment Type Duration Evolution ofof DM Classification of the Identif. Age Sex DM* evolution of the lesionlesion Type of lesion HCJQ  65 y** F II 13 y Ischemic, no 33 days WagnerIV. distal pulses, Amputation of 0.05 ABI 5^(th) toe residual base OFW53 y M II 21 y Neuropathic 81 days Wagner IV. with calcifiedTransmetatarsal macroangiopathy amputation and BI 1.2 residual base JIFM61 y F II 16 y Ischemic with 21 days Wagner V. pulse absence Necrotizingand 0.5 ABI fasciitis in plantar and metatarsal areas of the left lowerextremity *DM: Diabetes mellitus, **y: years

The treatment followed with each of the patients is described below:

Patient HCJQ. Ischemic, without distal pulse and occluded from thepopliteal sector. The lesion is a lateral amputation residual base ofthe fifth toe, showing exposed capsules and tendon clearly in ischemicnecrosis. The lesion was classified as grade IV according to the Wagnerscale. Its size was 10×4 centimeters. Treatment is established, when, onthe sixth day of amputation, the amputation residual base was found inclinical atony and cyanosis, progressing in this way for five days,despite all systemic and local pharmacological measures. Infiltrationswere initiated after surgical debridement of necrotic material,expecting improvement of the local microenvironment and promotion ofgranulation tissue development. This was first observed after the fifthinfiltration session. The patient received a total of 12 sessions ofinfiltration. The treatment favored the presence of productive andbleeding granulation tissue. Intensive centripetal epithelial migrationwas observed, although there was no notable contraction of the edges.After 38 days of treatment onset, the lesion was completelyepithelialized without requiring a graft. There were no acute or delayedadverse reactions. At 12 months, the lesion was free of localrecurrences.

Patient OFW. Neuropathic, 81 days of evolution with trans-metatarsalamputation residual base classifiable as Wagner IV at the time ofadmission. The size of the lesion is 14×7 square centimeters. Distalpulses are perceived with an ABI of 0.9, despite showing signs ofdysesthesia. The treatment is established when, during 42 days of localcures and application of physiological saline compresses, the lesionshows very poor granulation response. Infiltrations begin and areperformed daily during the first week of treatment to rescue the localcells. Subsequently, the schedule continues 2 times a week, for threeweeks. With this schedule, full productive and bleeding granulation wasachieved. The tissue was covered with a partial-thickness skin graftobtained from the contralateral anterior thigh. Similar to the patientpreviously described, the treatment was well tolerated and a year laterthe lesion was still epithelialized.

Patient JIFM. Female, former smoker, 61 years old, ABI of 0.4 in theleft lower extremity, shows necrotizing fasciitis in the metatarsal andplantar foot areas. The lesion is classified as grade V Wagner scale.The patient was treated surgically; excision of all necrotic tissueand/or contaminated soft tissue and bone was carried out. Systemicpolyvalent antibiotic therapy is established. After 48 hours of thesurgery, the first inspection of the lesion was made, local cleaningperformed, and infiltrative treatment with the vesicles is begun. Duringthe first 10 days, a treatment regimen of attack doses of EGF vesicleswas installed, which later could be reduced to two infiltration sessionsduring 5 weeks. The treatment helped save a foot without any othertherapeutic alternative. The patient has normal gait and satisfactorymotor command of the distal portion of the limb.

Example 18 Treatments Based on the Combined Application of Infiltrativeand Topical EGF Vesicles in Patients with Diabetic Foot Ulcers

In clinical cases, most of the treated lesions exceeded 90% probabilityof requiring amputation, according to the scale established by theUniversity of Texas. Treatments were administered combining the topicaland infiltrative routes. The general characteristics of the patientstreated correspond with those described in Example 16.

For the combined treatment, infiltration was used first, using theparenteral formulation comprising EGF vesicles (referred to in Example14). Afterwards, for the topical application, the spray described inExample 12 and the gel described in Example 13 were alternatively used.

The demographic characteristics of the treated population and thelesions are described in Table 14. In all cases treated the minor ormajor amputations were prevented.

TABLE 14 Demographic and epidemiological characteristics of the cohortof patients that received a combination therapy including infiltrationand topical treatment Type Duration Evolution of of DM Classification ofof the Identif. Age Sex DM* evolution the lesion lesion Type of lesionAFG  74 y** M II 22 y Ischemic, 20 days Wagner V popliteal Necrosis ofthe occlusive pattern, calcaneus soft ABI of 0.5 tissue and boneinvolvement LATR 61 y F II 18 y Ischemic - aortic 50 days Wagner IVbifemoral with Supracondylar macroangiopathy, amputation 0.4 ABIresidual base with ischemic plaque ZEM 57 y F II 22 y Pure neuropathic 7years Plantar extensive, granulated for 7 years with no evidence ofcontraction/ epithelialization JLHB 63 y M II 18 y Neuro-ischemic 7years Wagner IV Soft tissue abscess in the sole and inner- middlelateral region of the right lower extremity *DM: Diabetes mellitus, **y:years

Below, the treatment followed with each of the patients is described.

Patient AFG. Male with a long history of diabetes and history of pooradherence to treatments. The patient shows popliteal occlusion patternwith absence of distal pulses. The lesion debuts as a phlyctena that iscontaminated and rapidly advances to necrotize all soft tissues of thecalcaneus region. During a first surgical cure soft tissues are removed.Five days later, another surgery is necessary, which leads todevastation of bone material. Intensive polyvalent antibiotic therapy isestablished and 20 days after the first surgery, infiltrative treatmentwith EGF vesicles is started. A daily treatment session and local cureis established for the first two weeks. This is followed by cures andtreatment on alternate days using the topical gel formulation describedin Example 13 containing EGF:cetrimide:cholesterol vesicles preparedaccording to Example 3, on the edges and wound surface, during thefollowing 8 weeks. After 10 weeks of treatment, the lesion wascompletely epithelialized.

Patient LATR. Ischemic, lacking distal pulses and with macrovasculardisease of both lower limbs. ABI is 0.6, with no possibility ofrevascularization surgery due to calcifications. Prior contralateralamputation was performed three years ago. Has fine artery thrombosis inthe fourth and fifth toes, which were both amputated with extensive deeplateral wedge. Two weeks after surgery, the amputation residual baseshowed signs of atony and was refractory to granulation. Therefore inthe third week, treatment with the vesicles was started, initially byinfiltrative treatment with a dose between 25 and 125 μg of EGF perpoint of injection, deposited in the edges and bottom of the surgicalarea. This treatment modality is used until all cavities and tunnels arefilled with granulation tissue, which took place approximately afterthree weeks. Subsequently, it was decided to continue the treatmenttopically, by using gel with the composition described in Example 13,containing EGF:cetrimide:cholesterol vesicles. The treatment isperformed on the edges and wound surface, especially focusing on thedermo-epithelial border of the wounds to stimulate re-epithelialization.The treatment was performed on alternate days until completeepithelialization in 5 weeks.

Patient ZEM. Female, 57 years old, with Wagner grade III lesion andevidence of neuropathy, plantar lesion has granulated for 7 years, butthere is no evidence of contraction/epithelialization. After surgicalcleaning and stimulation of the edges, infiltrative treatment wascarried out with a dose of 75 μg of equivalent EGF per injection point,using the formulation described in Example 14 containingEGF:BKC:cholesterol vesicles, deposited in the edges of the surgicalarea. This treatment modality was applied during the first four weeks onalternate days. Subsequently, it was decided to continue using thetreatment on alternate days with the topical spray formulation describedin Example 12 containing EGF:CTAB:cholesterol vesicles. EGF vesicles areatomized on the edges and surface of the lesion; with special focus onthe dermo-epithelial border of the wounds to stimulatere-epithelialization, the treatment is performed on alternate days untilcomplete epithelialization in 8 weeks. The evolution of the healingprocess is shown in FIG. 7.

Patient JLHB. Neuropathic, developed an extensive infectious lesion asconsequence of a dry burn on the foot of the right lower extremity. Itfinally leads to transmetatarsal amputation, to which a 10-centimeterlateral wedge is added. Treatment is started with intravenousantibiotics and systematical local cures every 48 hours. The lesionshowed tendency to granulate very slowly and atony of the edges 20 daysafter surgery. Infiltrative treatment is begun with vesicles loaded with75 μg of EGF on alternate days in the residual base, as well as thewedge. By the third week of treatment the application starts, onalternate days, with the topical spray formulation described in Example12 containing EGF:CTAB:cholesterol vesicles, reaching the deep zone ofthe lateral wedge. The treatment combination accelerated totalgranulation and spontaneous epithelialization over an area of more than60 square centimeters.

Example 19 Demonstration of the Therapeutic Efficacy of the Use of EGFVesicles in a Lethal Model of Acute Lung Injury (ALI) or AdultRespiratory Distress Syndrome (ARDS) in Rats

Male Sprague Dawley rats with body weight between 250 and 280 grams wereused. Lung injury was induced under general anesthesia(ketamine/xylazine) by intratracheal instillation of a combination oflipopolysaccharide (LPS)-zymosan. Immediately afterwards, the animalswere randomly assigned to three experimental groups of 12 animals each.

-   Group A: treated with physiological saline.-   Group B: treated with an spray of EGF in physiological saline at a    concentration of 25 μg/ml/kg.-   Group C: treated with cholesterol:BKC:EGF vesicles described in    Example 4, with equivalent concentration of 25 μg/ml of EGF, and    administered as in Group B.

The animals were allowed to evolve without any treatment until the onsetof the first symptoms. Six hours after the application of LPS/zymosan,rats showed tachypnea associated with forced exhalation. At this point,the animals showed arterial PO₂ saturation of 65% and clear respiratoryacidosis.

Therefore, treatments were begun approximately 8 hours after the toxinswere instilled. The experiment was aimed at evaluating the effect oftreatment with both EGF formulations solely on the acute phase of ARDS.The treatments were performed by orofacial mask adapted from a volatileanesthesia machine. Treatments were performed twice daily. The animalswere also treated once a day with hydrocortisone acetate (10 mg/kg) ascoadjuvant treatment.

After 72 hours of treatment onset, the study was stopped. Survivinganimals were anesthetized and subjected to deep bronchoalveolar lavagein 5 ml of sterile saline for cytological and biochemical study, as wellas arterial blood sampling for determination of blood gas parameters.Then the lungs were insufflated with 10 ml of 10% neutral formalin andprocessed for histology.

Table 15 shows the daily mortality results per group. As it can be seen,the treatment with EGF nebulization controlled the progression of acutelung injury. Notably, the best effect in terms of survival was in thegroup that received EGF incorporated into vesicles. This suggests thatprolonged occupation of multiple EGF receptors exerts a more consistentpharmacological effect than EGF as particles in a simple liquid aerosolin physiological saline.

TABLE 15 Mortality in each group Group 24 hours 48 hours 72 hours Deathtotal A 3 6 1 10 (83.3%)  B — 5 — 5 (41.6%) C 2 — — 2 (16.6%)

The results of bronchoalveolar lavage confirmed the protection exertedby EGF to the alveolar wall and septal capillary endothelium.Furthermore, it was observed that the protection afforded by thetreatment with EGF incorporated into vesicles includes improvedventilation capacity expressed in greater arterial PO₂ rate, and keepingarterial pH very close to normal. These data are shown in Table 16.

TABLE 16 Results of bronchoalveolar lavage and lung function Total phos-Inflamma- Red blood pholipids tory cells cells per g of ArterialArterial Group per ml per ml lung tissue saturation pH A 3.0 × 10⁴ 2.0 ×10³ 188.6 μg 51.8 ± 2.2 6.58 ± 0.4 B 2.2 × 10³ 5.0 × 10² 241.0 μg  76 ±8.3 7.12 ± 0.1 C 0.5 × 10³ — 1.32 mg 91.7 ± 5.6 7.33 ± 0.2

Histological analysis of the lungs from each group showed that EGFtreatment, particularly in the group receiving the vesicle formulationprotects the lung parenchyma. This is demonstrated by the presence ofrelative preservation of the alveolus/septum ratio, reduction of theseptal wall permeability edema, as well as the presence of hemorrhagicfoci and eosinophilic material in the alveolar lumen. Less inflammatoryreaction was found in the animals treated with the formulation ofvesicle-incorporated EGF, compared to those receiving physiologicalsaline solution. The experiment allows us to infer that interventionwith cholesterol:BKC:EGF vesicles, described in Example 4, exerts moreintense/prolonged pharmacological effect on the lung parenchyma exposedto known inducers of ALI. The protection conferred covers not onlyimprovement of the structural integrity of the lung, but also presentssubstantial correction of functional parameters.

Example 20 Compassionate Treatment with EGF Vesicles of a Critically IllPatient with Adult Respiratory Distress Syndrome (ARDS)

Description of the case: Patient AGJ, male, 75 years old, withoutpathological medical history, who suffers abdominal trauma progressingto septic shock. ARDS occurs as an evolutionary complication, leading toventilation difficulty with dyspnea and cyanosis refractory to oxygentherapy, low saturation (11 mm Hg) and blood pH changes. A radiologicalimage shows cotton wool spots in the parenchyma of both lungs. It wasdecided to start treatment with positive end-expiratory pressure andother routine drugs. In order to protect the lung parenchyma in theacute exudative phase, cholesterol:BKC:EGF vesicles described in Example4 are administered at a dose of 200 μg equivalents of EGF per liter ofmedical oxygen, twice daily. The measures allowed a favorable andprogressive evolution of the patient to normal, preventing the fibrosingalveolitis phase. On the third day of treatment, positive pressureventilation could be suspended, and on the fifth day the cotton-likeimage had been completely cleared.

Example 21 Demonstration of the Therapeutic Efficacy of EGF-Vesicle Usein Patients with Distal Left Ulcerative Colitis

This is a cohort of 8 patients with distal ulcerative colitis diagnosis,after several colonoscopies and biopsies were performed. Patientsreceived a special medical diet, as well as pharmacological treatmentwith azulfidine or sulfasalazine. Because this cohort had remainedclinically active for more than 12 months and refractory to allpharmacological interventions, including corticoids, they are evaluatedfor compassionate treatment by low enema administration ofcholesterol:cetrimide:EGF, vesicles described in Example 3. For this,cholesterol:cetrimide:EGF vesicles are administered in physiologicalsaline, at an equivalent EGF concentration of 50 μg/ml, in a volume of20 ml. The enemas were applied in left lateral decubitus, every nightbefore sleeping time. At 7 days after onset of the enemas associatedwith other previous medical measures, melenas, pain, and generalsystemic malaise were eradicated. After 10 days of treatment, acolonoscopy is performed, detecting substantial reduction of mucosalinflammation, healing of most lesions and significant reduction in St.Mark's index. Treatment was discontinued 21 days after onset, whencolonoscopies are repeated, confirming the effect of healing andreduction of inflammation brought about by the treatment with enemascontaining the cholesterol:cetrimide:EGF vesicles. Biopsies taken fromthe 8 patients, at 21 days of starting treatment, showed thedisappearance of cryptitis.

Example 22 Compassionate Use of EGF Vesicles to Revert Mucosal LesionsCaused by Cancer Chemotherapy

Patient QED, male, 32 years old, who developed hemorrhagic mucositis inthe bladder and lower parts of the urinary tract, as consequence ofcyclophosphamide and busulfan therapy, after bone marrowtransplantation. After 72 hours with hematuria and substantialproteinuria (400 mg/24 hours), the patient received, together withgeneral medical support measures, a cystoclysis through which aninfusion of physiological saline solution containingcholesterol:CTAB:EGF vesicles, prepared as in Example 2 at equivalentconcentration of 150 μg/mL of EGF. At the end of the first 24 hours,only traces of red blood cells were detected in urine and proteinuria of170 mg/24 hours.

Patient IRT, female, 27 years old, develops mucositis of upper parts ofthe gastrointestinal tract, in the course of polychemotherapy fortreatment of non-Hodgkin lymphoma. A full-blown clinical picture,including aphthous manifestations, sialorrhea, even hematemesis,abdominal distension, general malaise, fever and other symptoms appears.The process begins to be managed by pharmacological measures, whichincluded proton pump inhibitors, mucoprotectors, restitution of volemiaand analgesic parenteral opioids. Twenty-four hours after onset of theclinical picture, the patient receives oral applications of a salinesolution containing cholesterol:cetrimide:EGF vesicles, prepared as inExample 3 at an equivalent concentration of EGF of 50 μg/mL. Using anasogastric catheter, a volume of 250 ml of this solution was instilledevery four hours. Hematemesis ceased after 24 hours of gastricirrigation onset, as well as the distension and acute paresis.Similarly, the need for volume support and other management measures wasprogressively reduced.

Example 23 Synthesis of β-Sitosterol:CTAB:EGF Vesicles Using CompressedFluid Technology

These vesicles were similarly synthesized to those in Example 2, but inthis case, on one hand, a solution of 77.71 mg of β-Sitosterol in 2.88mL of ethanol and; on the other one, an aqueous solution of 2.83 mg/mLof CTAB and EGF, to the desired concentration, was used.CTAB:β-Sitosterol (1:1) vesicles were obtained with EGF incorporated ata concentration of 5 μM. The results for physical appearance, mean sizeand Z-potential are shown in Table 17. It can be observed that thevesicle preparation is stable and has a relatively small mean size andPDI. Mean size, particle size distribution and the Z-potential weredetermined by DLS. Furthermore, similar to the vesicle preparationsshown in the previous examples, which comprise in their composition acationic surfactant of quaternary ammonium type, they have positiveZ-potential values much higher than +30 mV, which predicts their highlong-term stability. FIG. 3D shows that spheroidal shapes withunilamellar structure predominate, according to the vesicle morphologystudy performed by cryo-TEM. These vesicles also have features that makethem very attractive from a pharmaceutical point of view.

TABLE 17 Physical appearance, mean particle size and Z-potential of theβ-Sitosterol:CTAB:EGF vesicles at a given composition. Composition(EGF:β-Sitos- Mean size Z-Potencial terol) Physical appearance (nm)(PDI)* (mV) (±SD)* 5 μM:1M Disperse opalescent 218.8 (0.277) +78.6(±12.0) solution *Nano-ZS (Malvern Instruments, United Kingdom),PDI—polydispersity index

The invention claimed is:
 1. Process for the preparation of vesiclescomprising epidermal growth factor (EGF), a cationic surfactant andcholesterol or derivatives of cholesterol, comprising: a) preparing anaqueous solution of EGF and a cationic surfactant, b) dissolvingcholesterol or derivatives thereof in an organic solvent to form anorganic solution, c) expanding the organic solution with a compressedfluid to form an expanded organic solution, and d) depressurizing theexpanded organic solution resulting from stage (c) on the aqueoussolution resulting from stage (a), wherein vesicles are prepared.
 2. Theprocess of claim 1, wherein the cationic surfactant is of quaternaryammonium type.
 3. The process of claim 1, wherein the organic solvent ofstage b) is a solvent selected from the group consisting of monohydricalcohols, polyhydric alcohols, ketones, ethylene diamine, acetonitrile,ethyl acetate and mixtures thereof.
 4. The process of claim 1, whereinthe compressed fluid is a compound selected from the group consisting ofcarbon dioxide, ethane, propane, hydrochlorofluorocarbons, andhydrofluorocarbons.
 5. The process of claim 1, wherein the ratio betweenthe quantity of compressed fluid and organic solvent corresponds to anapproximate molar fraction of compressed fluid of 0.3 to 0.95.
 6. Theprocess of claim 1, wherein step (c) is carried out in a reactor at anapproximate pressure of 1 to 30 MPa and an approximate temperature ofabout 10 to 70° C.
 7. The process of claim 1, wherein the EGF isdissolved in an aqueous solution containing a cationic surfactant with aconcentration above its critical micellar concentration.
 8. A processfor the manufacture of a cosmetic composition comprising incorporatingvesicles produced by the method of claim 1 into an acceptable excipient,wherein the cosmetic composition is for inhibiting senescence of theskin.